The long term goal of this research is to use H3N8 influenza virus as a model to determine viral genetic mutations in single or multiple gene segments of influenza virus A that facilitate interspecies transmission and induction of severe pulmonary disease. The hypothesis being pursued in these studies is: Transmission of equine influenza virus to dogs and adaptation in the new host to induce severe respiratory disease was characterized by mutations in one or several gene segments that allowed the virus to replicate to a greater extent in macrophages and induce higher levels of TNF-. The specific aims of this pilot research project are: 1)To compare virus replication and induction of TNF- mRNA and protein synthesis and other cytokines in canine alveolar macrophages following inoculation with H3N8 canine influenza viruses from 2003-2004 with those following inoculation with H3N8 equine influenza virus isolates ranging from 2003 to 1991; 2) To determine whether the canine influenza virus derived with a reverse genetics system (canine/FL/04-rg) replicates to similar extent and induces comparable levels of TNF- mRNA and protein in canine alveolar macrophages as wild type virus (A/canine/Florida/43/2004) does; and 3) To compare the capacity of canine and equine influenza virus isolates to replicate in primary airway epithelial cells and induce chemokines (IL-8 and MCP-2). The project will use primary isolated dog alveolar macrophages inoculated in vitro with equine influenza A viruses isolated from 1991 through 2003 and initial canine influenza isolates from 2003 and 2004. TNF- mRNA and protein will be measured with quantitative real-time PCR and antigen capture assays, respectively. Virus production will be measured by infectious plaque assay and virus matrix gene quantitative real-time PCR. PUBLIC HEALTH RELEVANCE: The long term goal of this research is to understand which genetic changes in influenza virus from horses allowed it to be able to infect dogs and cause serious respiratory disease that could be passed on to other dogs. Equine influenza viruses from 1991 through 2003 will be compared with canine influenza virus first identified in 2003-2004 for their ability to induce a protein (tumor necrosis factor or TNF) when lung cells become infected with virus. Increased tumor necrosis factor secretion by lung cells is believed to cause severe changes in the lung that result in death. Information from this study will be used in other studies to identify the specific gene changes that allowed the virus to become deadly in dogs. The information will help better understand how influenza viruses from animals evolve to become infectious to other animals and man and cause disease.